Novel protein having hemolytic activity and gene encoding the protein

ABSTRACT

Novel proteins having hemolytic activity giving the new approach of development of the drugs, and having the following property are provided:  
     (1) having hemolytic activity;  
     (2) having a molecular weight of about 50,000 Da (determined by SDS gel electrophoresis); and  
     (3) having an amino acid sequence represented by any of SEQ ID NO 1 and SEQ ID NO 2 as a partial amino acid sequence represented by the following amino acid sequences (1) and (2);  
     Amino acid sequence (1):  
     Tyr-Arg-Asp-Gln-Glu-Leu-Glu-Asp-Asn-Val-Lys  
     Amino acid sequence (2):  
     Lys-Trp-Pro-Asp-Tyr-Phe-Val-Tyr-Met-Glu-Ser-Ser-Ala-His-Gly-Tyr-Ile-Arg  
     (wherein, an amino acidresidue is written by the 3 letters notation defined by IUPAC and IUB) obtained from nematocyst of  Carybdea alata.

TECHNICAL FIELD

[0001] The present invention relates to novel proteins having ahemolytic activity and genes encoding thereof. More specifically, thepresent invention relates to novel proteins having the hemolyticactivity, a process for producing thereof, reagents, pesticides andmedicines utilizing physiological activities thereof.

BACKGROUND ART

[0002] The sting injury by the jellyfish in sea bathing has occurred invarious parts of the world. The sting injury by Carybdea rastonii orPhysalia physalis has also occurred frequently in Japan every year inthe season of sea bathing of the summertime. The degree of the symptomby sting differs by species of a jellyfish and the individualdifferences of patients. The first symptom is dermotoses, such as pain,flare, papule, vesicle and so on in the sting site. In a seriousillness, patients may die with generating of hemorrhagic maculae and thenecrosis, and also constitutional symptom, such as headache, high fever,nausea, dyspnea, and the fluctuation of a pulse. Although such stinginjury is occurring frequently, the determination and pharmacologicalproperties of the toxic components of jellyfish have not been studiedintensively. Therefore, the development of medicines for treatment ofthe sting by the jellyfish is hardly performed before the presentinvention.

[0003] For example, the serious sting injury by Carybdea alata in Hawaiior other places has been reported (R. H. Tamanaha et al., J. Am. Acad.Dermatol., 1996, 35, 991-993); however, the determination andpharmacological properties of the toxic components of this jellyfishhave not been studied. On the contrary, the studies on the toxiccomponents of Carybdea rastonii, which is family relation to Carybdeaalata, have well studied, and chemical and physiological properties ofthe toxic component of Carybde arastonii have been clarified (AkihikoSato, “Research on the toxic component of Carybdea rastonii”, TheJournal of the Ochanomizu Medico-dental Society, vol. 33, No. 2,131-151, June, 1985; International Laid-open Patent Publication No.WO99/50294).

[0004] On the one hand, since the known poisons from the nematocyst of ajellyfish were non-dialyzable high polymer and deactivated by treatmentwith acid or alkali, or by heating processing, organic solventprocessing, protease processing, etc., it was thought that the maincomponents of poison were proteins.

[0005] Moreover, the purification of the protein toxin derived from ajellyfish has also been tried; however, the isolation and thepurification of the active components maintaining the hemolytic activitywere not performed since the toxin of a jellyfish itself was very easyto be deactivated. Therefore, the physical and chemical properties ofthe toxin from jellyfish have never been clarified up to now. However,the method for isolation and purification of the unstable toxiccomponents of jellyfish in good yields has reported recently in theInternational Laid-open Patent Publication No. WO99/50294.

[0006] The detailed studies on the toxic component of a jellyfish isvery important for the development of drugs applying their variousphysiological activities, in particular, specific hemolytic activity andthe cytotoxic effect, and for the development of medicines for treatmentof the sting injury by the jellyfish.

[0007] Therefore, the problems to be solved by the present invention isproviding an approach to development of the drugs for treatment of thesting injury by the jellyfish by means of isolating the proteins orpeptides having as potent hemolytic activity as possible, in the statewhere the physiologic activity is retained. The present inventionfurther provides the approach to study similarities on embryology orstructure, and the species specificity of the protein having hemolyticactivity to evaluate the structure-activity relationship thereof.

DISCLOSURE OF THE INVENTION

[0008] The inventors extensively performed the research for isolatingthe proteins having the hemolytic activity from the nematocyst ofCarybdea alata using the hemolytic activity as the parameter, whileretaining these hemolytic activities. As the result, they found out theprocess for isolating and purifying the proteins retaining hemolyticactivities, and clarified the protein from Carybdea alata having thepartial chemical structure consisting the following amino acid sequences(1) and (2), and the molecular weight of about 50,000 Da (determined bySDS gel electrophoresis).

[0009] Amino acid sequence (1):Tyr-Arg-Asp-Gln-Glu-Leu-Glu-Asp-Asn-Val-Lys

[0010] Amino acid sequence (2):Lys-Trp-Pro-Asp-Tyr-Phe-Val-Tyr-Met-Glu-Ser-Ser- Ala-His-Gly-Tyr-Ile-Arg

[0011] (wherein, an amino acidresidue is written by the 3 lettersnotation defined by IUPAC and IUB)

[0012] Furthermore, they prepared the primers based on their partialchemical structures of the protein, and analyzed the gene sequence ofabout 900 base pair of said protein by conducting the RT-PCR to totalRNA prepared from the tentacle of Carybdea alata by using these primers.Consequently, they further determined the full primary amino acidsequence of the hemolytic active protein of Carybdea alata by means ofanalyzing the gene sequence in 5′-end and 3′-end using the 5′ RACEmethod and 3′ RACE method.

[0013] Therefore, one embodiment of the present invention provides thespecific protein having the amino acid sequence represented by SEQ ID NO3, or the amino acid sequence modified by the addition and deletion ofone or more amino acid, and/or the substitution by other amino acid tosaid amino acid sequence, with the above-mentioned physiological,physical and chemical properties.

[0014] Another embodiment of the present invention also provides theprocess for preparing such proteins.

[0015] Furthermore, another embodiment provides the gene encoding suchproteins, the process for preparing the specific proteins using thegene, and the drugs or the pesticides using the same.

[0016] The present invention further provides the pharmaceuticalcompositions containing the proteins using these properties,particularly, the pharmaceutical compositions having the plateletagglutination effect etc., or the pesticides utilizing the cytotoxicproperties of the proteins.

[0017] Moreover, since a specific antibody can also be obtained fromthis hemolytic active protein according to a conventional method (CellTechnology, separate volume, “Experimental protocol of antipeptideantibody”, Shujunsha Co.), the present invention also provides thepharmaceutical compositions containing said antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] The isolation and purification of the proteins having thespecific physiological activity provided by the present invention canspecifically be performed as follows. For example, the ultrasonicationof the nematocyst of Carybdea alata is carried out in phosphoric acidbuffer solution, and then supernatants are collected by the centrifugalseparation to obtain a crude extract. The object proteins can beseparated and purified by subjecting this crude extract to ion exchangehigh performance liquid chromatography using an ion exchange column suchas TSK-GEL (Toso Co.), and the gel filtration high performance liquidchromatography with a gel filtrate column such as Superdex-75 (PharmaciaCo.).

[0019] The structure of the protein provided according to the presentinvention obtained in this way can be determined by combining theanalysis procedure of the amino acid sequence by the selectivedegradation using the enzyme, and the analysis procedure of a genesequence using the PCR method etc. For example, the amino acid sequencecan be determined by processing the protein separated and purified asmentioned above with a lysyl-endopeptidase, fractionating the fragmentusing a high performance liquid chromatography, and analyzing it usingan amino acid sequencer etc. Next, the gene sequence of the proteins canbe determined by RT-PCR method etc. using the primers prepared on thebasis of the amino acid sequence. Finally, the full primary amino acidsequence of the proteins can be clarified by determining the amino acidsequence on the basis of the gene sequence.

[0020] It was confirmed by such analysis that the protein providedaccording to the present invention has the molecular weight of about50,000 Da (measured by SDS gel electrophoresis), and the partial aminoacid sequences have the above-mentioned amino acid sequences (1) and(2).

[0021] As a result of a homology search on the partial amino acidsequences, except for exhibiting about 40% of homology between theprotein of the present invention and the hemolytic active proteins fromCarybdea rastonii, which is family relation to Carybdea alata, thehomology between the protein of the present invention and the knownother proteins was very low. Therefore, it was suggested that theprotein of the present invention having the hemolytic activity iscompletely novel protein, which is not similar to the known proteins.

[0022] Next, the determination of the gene sequence of about 1,000 basepairs by performing RT-PCR to total RNA prepared from the tentacle ofCarybdea alata using the primers prepared on the basis of the partialamino acid sequence, and the determination of the gene sequences of the5′-end and the 3′-end using the 5′ RACE method and 3′ RACE method wereperformed. Consequently, it is concluded that the hemolytic activeprotein of Carybdea alata has the full primary amino acid sequencerepresented by SEQ ID NO 3, and the gene encoding thereof has the basesequence represented by SEQ ID NO 4.

[0023] The method for preparing the specific protein of the presentinvention by separation and purification is characterized in retainingthe hemolytic activity. For example, the separation and the purificationin the state of retaining such hemolytic activity are attained byperforming the processing such as ultrasonication using theabove-mentioned phosphoric acid buffer solution or various highperformance liquid chromatography in 10 mM phosphoric acid buffersolution (pH 6.0) containing above 0.1 M NaCl, preferably above 0.3 M,and more preferably above 0.5 M, at below 10° C., preferably below 5° C.

[0024] Therefore, the present invention also provides the method forpreparing the protein by extracting and purifying them from thenematocyst of the Carybdea alata in the state of retaining thephysiological activity.

[0025] The specific protein of the present invention also can beprepared by the gene recombination method. Preparation by the generecombination method can be performed according to a conventionalmethod. For example, it can be obtained by preparing the vectorintegrated with the gene represented by SEQ ID NO 4, transforming a hostcell by the vector, incubating or growing the host cell, and isolatingand purifying the proteins having hemolytic activity of interest fromthe host cell or culture solution.

[0026] Since the protein provided according to the present invention hasa hemolytic activity, for example, it may be used for the medicamentshaving the cytolysis effect and for the reagents for research on ahemolysis. Furthermore, it provides the new approach for the developmentof drugs, such as a drug for treating the sting by the jellyfish, anddevelopment of pesticides, such as an insecticide, using the hemolyticor cytotoxic activity.

EXAMPLES

[0027] The present invention will be described in detail with referenceto the following examples; however, the present invention is not limitedto the examples.

Example 1

[0028] 1) Extraction of the Nematocyst of Carybdea alata

[0029] 200 mg of the nematocyst of the Carybdea alata obtained on theWaikiki Beach, Hawaii and cryopreservated at −80° C. was immersed in 8ml of 10 mM phosphoric acid buffer solution (pH 6.0), and treated for 15minutes by the ultrasonic wave (ultrasonic cleaner VS150, Iuchi Co.).The supernatant fluids were collected by centrifugal separation (3,000rpm, for 20 minutes). This operation was performed 3 times in total.Furthermore, the same extraction operation was repeated 3 times with 8ml of 10 mM phosphoric acid buffer solutions (pH 6.0) containing 1 MNaCl, and then all the supernatant fluids were collected. After theextraction operation, ion exchange HPLC (high performance liquidchromatography) of the following purification step was immediatelyperformed.

[0030] 2) The Purification by Ion Exchange HPLC (Column: TSK-GEL CM650S,Column Size: 20×220 mm, Toso Co.)

[0031] The above-mentioned column was equilibrated with 10 mM phosphoricacid buffer solution (pH 6.0) containing 0.3 M NaCl. After theequilibration, the supernatant fluids obtained by extraction in theoperation of the above-mentioned 1) were combined and diluted with 10 mMphosphoric acid buffer solution (pH 6.0) to 4 times. The solution wasloaded onto the above-mentioned column at a flow rate of the 3 ml/min.The column was washed with 100 ml of 10 mM phosphoric acid buffersolutions (pH 6.0) after the sample application. The elution was carriedout by the 60 minutes gradient in 0 to 0.7 M NaCl concentration (in 10mM phosphoric acid buffer solution: pH 6.0). Hemolytic activity wasshowed in many fractions eluting between 45 and 65 minutes after startof the gradient. In addition, hemolytic activity was examined about thehemolytic effect to sheep hemocytes (see the after-mentioned example 2).

[0032] 3) The Purification by Ion Exchange HPLC (Column: TSK-GEL CM5PW,Column Size: 7.5×75 mm, Toso Co.)

[0033] The above-mentioned column was well equilibrated with 10 mMphosphoric acid buffer solution (pH 6.0) containing 0.3 M NaCl. Thehemolytic active fractions obtained by purifying operation of theabove-mentioned 2) were diluted with 10 mM phosphoric acid buffersolution (pH 6.0) to 4 times. The solution was loaded onto theabove-mentioned column at the flow rate of 2 ml/min. The column waswashed with 30 ml of 10 mM phosphoric acid buffer solutions (pH 6.0)after the sample application. After washing, the elution was performedby the 60 min gradient in 0 to 0.8 M NaCl concentration (in 10 mMphosphoric acid buffer solution: pH6.0). Fractions having hemolyticactivity were eluted between 25 and 35 minutes after start of thegradient, and each fraction was applied to SDS-PAGE. The separatingcondition of the active component was verified, and the portionsseparated well were collected and used in the next step. On thecontrary, the portions not separated were further performed bychromatography to complete the separation of the active component.

[0034] 4) Concentration of the Hemolytic Active Component by IonExchange HPLC (Column: TSK-GEL CM5PW, Column Size: 7.5×75 mm, Toso Co.)

[0035] The column was well equilibrated with 10 mM phosphoric acidbuffer solution (pH 6.0) containing 0.3 M NaCl. The hemolytic activefractions obtained by purifying operation of above-mentioned 3) werediluted with 10 mM phosphoric acid buffer solution (pH 6.0) to 4 times.The solution was loaded onto the above-mentioned column at the flow rateof 2 ml/min. The column was washed with 30 ml of 10 mM phosphoric acidbuffer solutions (pH 6.0) after the sample application. After washing,10 mM phosphoric acid buffer solution (pH 6.0) containing 0.8 M NaCl wasthen rinsed and the sample adhered into the column was allowed to elute.In about 5 minutes after exchange of the solvent, the portion of thehemolytic active component condensed and eluted at a stretch wascollected.

[0036] 5) The Purification by Gel Filtration HPLC (Column: Superdex-75,Column Size: 16×600 mm, Pharmacia Co.)

[0037] Every 0.5-1.0 ml of the sample condensed by ion exchange HPLC ofabove 4) was applied to the above-mentioned column equilibrated with 10mM phosphoric acid buffer solution (pH 6.0) containing 0.8 M NaCl, andallowed to elute at the flow rate of 1 ml/min. Potent hemolytic activitywas found out in the fraction eluting between 50 and 60 minutes afterinjection of the sample. After confirming the separating condition bySDS PAGE, the protein of the present invention, a hemolytic toxin, wasseparated by collecting the active fractions (about 10 μg).

Example 2

[0038] Measurement of the Hemolytic Activity

[0039] Measurement of the hemolytic activity in each purification stepin the above-mentioned Example 1 and measurement of the hemolyticactivity of the protein of the present invention finally obtained wereperformed as follows.

[0040] 1) Method

[0041] Hemolytic activity was measured by hemolysis to a sheeperythrocyte. That is, every 200 μl of PBS(+) buffer solution containing0.8% of sheep erythrocyte was put into the microwell plates of 96 wells(round bottom type). 10 μl of the solution dissolved the fractionobtained in each purification step of the above-mentioned Example 1 in10 mM phosphoric acid buffer solution (pH 6.0) was added to the plate.It was allowed to stand at room temperature for 3 hours, and thehemolytic condition of the sheep erythrocyte of each plate was observed.In addition, the presence or absence of the retention of the hemolyticactivity was determined by whether the fraction obtained in eachpurification step exhibits a perfect hemolysis.

[0042] 2) Results

[0043] 2-1) The fraction obtained in each purification step of theabove-mentioned Example 1 exhibited the perfect hemolysis to the sheeperythrocyte, and therefore, it became clear that it retains thehemolytic activity.

[0044] 2-2) Moreover, the protein of the present invention having thehemolytic activity finally obtained by purification operation of theabove-mentioned 5) in Example 1 caused the perfect hemolysis to thesheep erythrocyte in the concentration below 100 ng/ml (about 2 nM).

Example 3

[0045] Determination of the Molecular Weight and the Partial Structureon the Proteins

[0046] 3-1) Determination of the Molecular Weight

[0047] The single band visualized by applying the protein of the presentinvention having the hemolytic activity obtained by purificationoperation of 5) in Example 1 to SDS gel electrophoresis (SDS-PAGE)according to the conventional method was compared with the proteinmolecular-weight marker (Pharmacia Co.). As the result, it wasidentified that the molecular weight of the protein of the presentinvention are about 50,000 Da.

[0048] 3-2) Decomposition with the Lysylendopeptidase

[0049] The protein was decomposed by adding 3 pM of AchromobacterProtease I (derived from Achromobacter lyticus M497-1: Takara Shuzo Co.)to 10 μg of protein according to the present invention having thehemolytic activity obtained by purification operation of theabove-mentioned 5) in Example 1, and incubating in 10 mM of Tris-HClbuffer solution (pH 9.0) at 30° C. for 20 hours. The protein digestedwith the enzyme was applied to the high performance liquidchromatography (column: Bakerbond wide pore ODS), and separated with the60 min gradient in 10 to 62% of acetonitrile concentration (in watercontaining 0.1% of trifluoroacetic acid) at the flow rate of 0.7 ml/min.Consequently, two peptide fragments eluting respectively at a retentiontime 25 and 49 minutes were obtained.

[0050] 3-3) Determination of the Amino Acid Sequence of Each Fragmentsby the Amino Acid Sequencer

[0051] The amino acid sequence of three peptide fragments obtained asmentioned above was determined according to the conventional methodusing Shimadzu PSQ-1 protein sequencer (Shimadzu Co.).

[0052] As the result, two fragments have the following amino acidsequences (1) and (2), respectively:

[0053] Amino acid sequence (1):Tyr-Arg-Asp-Gln-Glu-Leu-Glu-Asp-Asn-Val-Lys

[0054] Amino acid sequence (2):Lys-Trp-Pro-Asp-Tyr-Phe-Val-Tyr-Met-Glu-Ser-Ser- Ala-His-Gly-Tyr-Ile-Arg

[0055] (wherein, an amino acid residue is written by the 3 lettersnotation defined by IUPAC and IUB).

[0056] The homology search about each fragment with which the amino acidsequence was determined as mentioned above exhibited that the homologybetween these fragments and the known proteins was very low, except forexhibiting about 40% homology with hemolytic active proteins fromCarybdea rastonii. Therefore, it was suggested that the specific proteinof the present invention fractionated from the nematocyst of Carybdeaalata while retaining the hemolytic activity is completely novelprotein.

Example 4

[0057] Determination of the Full Amino Acid Sequence of the Protein andthe Gene Encoding the Amino Acids

[0058] 4-1) Preparation of Total RNA of Carybdea alata

[0059] The tentacle (about 0.5 g in wet weights) of Carybdea alata wascrushed in the liquid nitrogen, and homogenized in 5 ml TRIzol(registered trademark) reagent (GIBCO BRL Co.). To this mixture wasadded 1 ml of chloroform, and the mixture was agitated, and centrifugedwith the cooling centrifuge (Sakuma Co.) [13,000 rpm, for 15 minutes, at4° C.]. The upper aqueous layer was fractionated, and to this solutionwas added 2.5 ml of isopropanol, then, the mixture was allowed to standat room temperature for 10 minutes. The supernatant fluid was removedafter the centrifugal separation (13,000 rpm, for 10 minutes, at 4° C.)using the cooling centrifuge, and then 5 ml of 75% ethanol was added theresidue. The supernatant fluid was removed after the centrifuge (10,000rpm, for 5 minutes, at 4° C.) to obtain the residue, then, theair-drying of the residue was performed for about 10 minutes. 100 μl ofRNase-free water was added to the resulting residue, and the mixture wasincubated for 10 minutes at 60° C. to lyse RNA. About 0.5 mg of totalRNA was obtained according to the above-mentioned method.

[0060] 4-2) Cloning of a Partial cDNA

[0061] On the basis of amino acid sequence (1) and amino acid sequence(2), the following degenerate primers were designed and synthesized bythe conventional method: F-primer; GAY CAR GAR YTI GAR GAY AA R-primer;ATR TAI CCR TGI GCI SWI SWY TCC

[0062] (wherein, the above-mentioned alphabetic character was writtenbased on the “Nucleotide Abbreviation List” (Cell Technology, separatevolume, “Biotechnology Experiment Illustrated”: Shujunsha Co.).

[0063] Next, according to the following procedure, single-strand cDNAwas synthesized using SUPERSCRIPT (registered trademark)Preamplification System for First Strand cDNA Synthesis. That is, 1 μgof total RNA, oligo(dT)₁₂₋₁₈, and DEPC-treated water were mixed, and themixture was allowed to stand for 10 minutes at 70° C. Then, PCR buffer,25 mM MgCl₂, 10 mM dNTP mix, and 0.1 M DTT were added to this mixture,and the resulting mixture was pre-incubated for 5 minutes at 42° C.Superscript II RT (200 units/μl) was added to this mixture, and themixture was incubated for 50 minutes at 42° C. and for 15 minutes at 70°C. The RNase H was added to the mixture, and then, the resulting mixturewas incubated for 20 minutes at 37° C. to obtain 1st-strand cDNA.

[0064] Subsequently, according to the following conditions, PCR wasperformed using GeneAmp PCR System 2400 thermal cycler (Perkin-ElmerCo.). That is, 1st-strand cDNA, PCR buffer, dNTP mix, F-primer,R-primer, TaKaRa Ex Taq (registered trademark, Takara Shuzo Co.), andwater were mixed. The reaction was performed by heating the mixture at94° C. for 5 minutes, then repeating 3 cycles of 30 seconds at 94° C.,30 seconds at 45° C. and 2 minutes at 72° C., and further 27 cycles of30 seconds at 94° C., 30 seconds at 55° C. and 2 minutes at 72° C. Thereactant was then treated for 5 minutes at 72° C.

[0065] The obtained reaction solution was electrophoresed on 0.8%agarose gel to confirm the amplified PCR product in the combination ofF-primer and R-primer. The size of the PCR product was about 900 bp.

[0066] 4-3) Sequencing of the Partial cDNA

[0067] The PCR product was inserted into TA cloning vector pCR2.1(Invitrogene Co.), and the recombinant was transformed to theEscherichia coli JM109. The transformant was cultured on LB (containing50 μg/μl of ampicillin) agar medium. According to the followingconditions, colony PCR was performed to the colonies obtained as atemplate using the M13 universal primer. The strain of Escherichia coli,PCR buffer, dNTP mix, M13 FW primer, M13 RV primer, TaKaRa Ex Taq(registered trademark, Takara Shuzo Co.), and water were mixed. Thereaction was performed by heating the mixture at 90° C. for 10 minutes,then repeating 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C.and 2 minutes at 72° C., and further heating at 72° C. for 5 minutes.The reaction solution was electrophoresed on 0.8% agarose gel and thetarget colony PCR product was purified on the spin column of MicroSpin(registered trademark) S-400 (Amersham Pharmacia Co.). Then, thesequencing of the obtained product was conducted by using ABI PRISM 310Genetic Analyzer (Applied Biosystems Co.).

[0068] The obtained sequence was analyzed using gene analysis soft wareGENETYX-MAC (Software Development Co.). As the result, the partial cDNAsequence of about 900 bp was analyzed.

[0069] 4-4) Sequencing of the Full-Length cDNA

[0070] Following primers were synthesized based on the base sequence ofthe partial cDNA: 5′-RACE-1R; ACA GCA TCT CTG ACC GAA TC 5′-RACE-2R; AATGGA CCT CGG TCA GAT TC 5′-RACE-3R; CAC TGT TGA CGA AGG TGA TG3′-RACE-1F; GGA ACA GCT GAT GAA GAT CC 3′-RACE-2F; GGT GAG CAA GGT TACTTC AC

[0071] Next, according to the following procedure, 5′ RACE and 3′ RACEwere performed using 5′/3′ RACE Kit (Boehringer Mannheim Co.).

[0072] (a) 5′ RACE

[0073] 1 μg of total RNA, cDNA synthesis buffer, dNTP mix, 5′-RACE-1R,AMV reverse transcriptase, and DEPC-treated water were mixed, and themixture was incubated for 60 minutes at 55° C. and for 10 minutes at 65°C. to obtain 1st-strand cDNA.

[0074] Next, 1st-strand cDNA thus obtained was purified on the spincolumn, then, reaction buffer and 2 mM dATP were added to the 1st-strandcDNA, and the mixture was allowed to stand for 3 minutes at 94° C.Terminal transferase (10 units/μl) was added to the mixture, and theresulting mixture was incubated for 20 minutes at 37° C. After theincubation, 1st-strand cDNA, PCR buffer, dNTPmix, 5′-RACE-2R,oligo(dT)-anchorprimer, and water were added to the above mixture. Thereaction was performed by heating the mixture at 94° C. for 5 minutes,then repeating 30 cycles of 30 seconds at 94° C., 30 seconds at 55° C.and 1 minute at 72° C., and further heating at 72° C. for 5 minutes.Consequently, the nested-PCR was performed to the 1st-PCR product as atemplate using the combination of 5′-RACE-3R and PCR anchor primer underthe same condition as 1st-PCR.

[0075] The 1st-PCR product and the nested-PCR product wereelectrophoresed on 1.5% agarose gel to confirm the band of about 600 bp.This nested-PCR product was inserted into TA cloning vector, and thesequencing was performed according to the determination of the basesequence of cDNA described in the above-mentioned 4-3), then thesequence was analyzed.

[0076] (b) 3′ RACE

[0077] 1 μg of total RNA, cDNA synthesis buffer, dNTP mix,oligo(dT)-anchor primer, AMV reverse transcriptase, and DEPC-treatedwater were mixed, and the mixture was incubated for 60 minutes at 55° C.Subsequently, the reactant was treated for 10 minutes at 65° C. toobtain 1st-strand cDNA.

[0078] Next, 1st-PCR thus obtained was performed under the followingcondition. 1st-strand cDNA, PCR buffer, dNTP mix, 3′-RACE-1F, PCR anchorprimer, TaKaRa Ex Taq (registered trademark, Takara Shuzo Co.), andwater were mixed. The reaction was performed by heating the mixture at94° C. for 5 minutes, then repeating 30 cycles of 30 seconds at 94° C.,30 seconds at 55° C. and 2 minutes at 72° C., and further heating at 72°C. for 5 minutes. The nested-PCR was performed to the 1st-PCR product asa template using the combination of 3′-RACE-2F and PCR anchor primerunder the same condition as 1st-PCR.

[0079] The 1st-PCR product and the nested-PCR product wereelectrophoresed on 1.5% agarose gel to confirm the band of about 600 bp.The nested-PCR product was inserted into TA cloning vector, thesequencing was performed according to the determination of the basesequence of cDNA described in the above-mentioned 4-3), and the sequencewas analyzed.

[0080] As a result, the size (2000 bp) and the sequence of cDNA encodingthe novel hemolytic active protein of Carybdea alata, and the number(463aa) and the sequence of amino acid of the protein became clear. Thatis, the hemolytic active protein of Carybdea alata has the amino acidsequence represented by SEQ ID NO 3, and the gene encoding thereof hasthe base sequence represented by SEQ ID NO 4.

[0081] The novel protein of the present invention obtained as mentionedabove is the specific protein having the following physiologicalactivity, and physical and chemical property, as indicated by theexample:

[0082] (a) having hemolytic activity;

[0083] (b) having a molecular weight of about 50,000 Da (determined bySDS gel electrophoresis);

[0084] (c) having the amino acid sequences 1 and 2 described above as apartial amino acid sequence; and

[0085] (d) having the amino acid sequence represented by SEQ ID NO 3 asthe full amino acid sequence.

Industrial Applicability

[0086] Since the protein having the hemolytic activity derived from thenematocyst of Carybdea alata provided according to the present inventionis a novel protein which is not similar to known protein, as a result ofthe homology search on the partial amino acid sequence and the fullprimary amino acid sequences, it is useful as a biochemical reagent forexample, elucidating the mechanism of a hemolysis etc.

[0087] It also provides the new approach directed to development ofdrugs, such as the medicine for treating the sting by the jellyfish, onthe basis of study of correlation of the structural activity in amolecular level, and the antibody on the protein or the partial peptide,etc. Furthermore, it is useful as the drugs having a plateletagglutination effect etc., and pesticides using a hemolytic activity.

1 12 1 11 PRT Carybdea alata 1 Tyr Arg Asp Gln Glu Leu Glu Asp Asn ValLys 1 5 10 2 18 PRT Carybdea alata 2 Lys Trp Pro Asp Tyr Phe Val Tyr MetGlu Ser Ser Ala His Gly Tyr 1 5 10 15 Ile Arg 3 463 PRT Carybdea alata 3Met Ser Arg Gly Tyr Ser Leu His Leu Val Leu Phe Leu Val Leu Ser 1 5 1015 Thr Ala Phe Pro Ser Gln Ala Arg Leu Ser Arg Tyr Arg Arg Ser Ala 20 2530 Ala Asp Ala Val Ser Thr Asp Ile Asp Gly Ile Ile Gly Gln Leu Asn 35 4045 Asp Leu Gly Thr Asp Thr Lys Arg Leu Lys Glu Ala Leu Gln Gly Val 50 5560 Gln Glu Ala Val Lys Lys Glu Pro Ala Thr Thr Ile Ala Lys Val Ser 65 7075 80 Thr Ile Val Gly Ser Val Gly Gly Ser Leu Ser Lys Phe Lys Ser Gly 8590 95 Asp Pro Phe Asp Val Ala Ser Gly Cys Leu Asp Ile Ile Ala Ser Val100 105 110 Ala Thr Thr Phe Gly Gly Pro Tyr Gly Ile Ala Ile Gly Ala ValAla 115 120 125 Ser Leu Ile Ser Ser Ile Leu Ser Leu Phe Ser Gly Asn SerMet Gly 130 135 140 Ser Ala Ile Lys Gln Val Ile Asp Asp Ala Phe Lys LysTyr Arg Asp 145 150 155 160 Gln Glu Leu Glu Asp Asn Val Lys Gly Ala LysArg Thr Phe Asn Ala 165 170 175 Val Ile Thr Phe Val Asn Ser Val Ser LysThr Glu Asn Leu Thr Glu 180 185 190 Val His Leu Asp Ser Val Arg Asp AlaVal Arg Val Asp Ala Phe Thr 195 200 205 Asn Met Leu Gly Val Leu Glu SerArg Ile Asn Arg Gly Ser Val Ser 210 215 220 Thr Asp Asn Asn Glu Ala MetArg Thr Ile Asn Phe Ile Phe Leu Tyr 225 230 235 240 Leu Gln Leu Ser ValMet Arg Glu Thr Leu Leu Thr Gln Val Ile Leu 245 250 255 Leu Tyr Lys ArgAla Gly Gly Ala Tyr Asp Glu Leu Ala Leu Ser Leu 260 265 270 Ser Leu ThrSer Asp Gln Asn Lys Glu Ala Thr Arg Glu Thr Val Thr 275 280 285 Phe LeuHis Gln Met Glu Thr Lys Tyr Ser Leu Cys Gly Ser Tyr Tyr 290 295 300 TyrPro Ile Asp His Ser Lys Ala Ala Ile Gly Ile Leu Lys Leu Thr 305 310 315320 Lys Phe Phe Gly Val Pro Asp Pro Ala Arg Tyr Thr Phe Asp Gly Leu 325330 335 Tyr Tyr Arg Met Gln Asn Arg Ala Trp Asn Arg Tyr Ser Ile Cys Lys340 345 350 Glu Ser Tyr Ala Gly Asn His Met Phe Arg Gly Cys Lys Asp SerSer 355 360 365 Tyr His Gly Ile Arg Ile Lys Lys Leu Glu Asn Gly Tyr HisThr Ile 370 375 380 Thr Leu Arg Ser Lys Ala Met Tyr Val Thr Lys His AlaGln Gly Trp 385 390 395 400 Gly Trp Gly Thr Ala Asp Glu Asp Pro Gly GluGln Gly Tyr Phe Thr 405 410 415 Phe Ile Pro Leu Thr Asn Gly Phe Tyr MetVal Ser Thr Lys Lys Trp 420 425 430 Pro Asp Tyr Phe Val Tyr Met Glu SerSer Ala His Gly Tyr Ile Arg 435 440 445 Ser Trp His Tyr Asn Pro Asp ProGln Gly Gln Trp Lys Ile Leu 450 455 460 4 2042 DNA Carybdea alata 4taaatggacc gtgtacaggc tcatctataa aaactattat ttgtgttttt aatttcaatt 60actagattca tcatgtctcg tggatatagc ttgcaccttg tgctttttct agttctttcc 120acagcattcc catctcaagc tagattatcg agatatcgtc gaagcgcagc cgatgccgta 180agcaccgata tcgacggcat cattggacag ctcaatgatc tcggtacaga taccaagcga 240ttaaaggaag ctctacaggg agttcaggaa gctgttaaaa aagagcccgc taccactatt 300gctaaagtat caactatcgt cgggtcagtt ggaggttcat tgagcaagtt caagtcagga 360gatccctttg atgttgcttc agggtgtctg gacatcattg ccagtgttgc tacaacattt 420ggaggtccat acgggattgc tattggggca gtagcatcat tgatttcctc tattcttagc 480ctcttctctg gaaatagtat gggaagtgca atcaaacaag ttattgacga cgctttcaag 540aaatatcgcg atcaagagtt ggaagacaat gtaaaaggag caaaaaggac ctttaatgcc 600gtcatcacct tcgtcaacag tgtatcaaag acagagaatc tgaccgaggt ccatttggat 660tcggtcagag atgctgttag agttgatgca tttaccaaca tgctaggtgt cttggagagc 720agaatcaatc gcggctctgt gtccaccgat aacaatgaag caatgagaac catcaatttc 780atcttcttgt acttacaact gtccgtgatg cgtgaaacac tgttgactca agttattctt 840ttgtacaagc gtgcgggtgg tgcatatgat gagctggcac tgtctctgtc cttaacaagt 900gatcaaaaca aggaagcgac aagagaaacg gttacgtttt tacatcaaat ggaaaccaag 960tattctctct gtggttccta ctactaccct attgaccact ctaaggcagc cattggtatt 1020cttaaactca caaaattttt tggagtgcca gatcctgcaa gatacacgtt tgatggtctt 1080tattacagaa tgcaaaacag ggcatggaat cggtatagca tctgtaaaga atcttatgcg 1140ggcaatcaca tgtttcgggg ctgcaaagat tcaagttacc atggaattag gatcaaaaag 1200ctggaaaatg gttaccatac tattaccctg agatcaaaag ccatgtatgt cacgaaacac 1260gctcaaggat ggggctgggg aacagctgat gaagatccag gtgagcaagg ttacttcact 1320ttcatccctt taacaaatgg tttttacatg gtttctacca agaagtggcc agattacttt 1380gtgtacatgg aaagcagtgc gcatggttat attcgaagct ggcattacaa ccctgatcca 1440cagggacaat ggaaaatctt gtaattgcta cggtgatttt tgaagttatc ccagataatg 1500actcagtcat agagcaatcg ttagagttgc ttcataatct aagttgcatt tctcgaacac 1560gcatgcgtcg gagttgctaa tacgtcctac gaacaggtgt attctgattt agtcctggtg 1620atttctttac ttgtattttt acgctattct caaagagttt caattttcga cgcagagact 1680aaatgtagat taaacgattt ttgtggtgtc aaagaaaagg aaaggaacag tgttgctttg 1740tagtaccacg gaaataaaag tgaaagacca ctcaatgcat tgttttcatg ctgaaaatag 1800aagtagtcac aactaacaaa cttcaattct aatatttcaa ttgtttattc atttgctctc 1860ttttcccttc caacaaccgc tgcaagtgat caatttatct acaagttagt aatcattaac 1920actatcgttg atcgtcctcg gataactcga atataattca tgcgtccagc gagtttgact 1980tatgtatctt agattgtgta gcatatcagc atatcaataa aatatcacaa aaaaaaaaaa 2040aa 2042 5 20 DNA Artificial Sequence Description of Artificial SequencePrimer 5 gaycargary tngargayaa 20 6 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 atrtanccrt gngcnswnsw ytcc24 7 18 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 7 tttttttttt tttttttt 18 8 20 DNA ArtificialSequence Description of Artificial Sequence Primer 8 acagcatctctgaccgaatc 20 9 20 DNA Artificial Sequence Description of ArtificialSequence Primer 9 aatggacctc ggtcagattc 20 10 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 10 cactgttgac gaaggtgatg 20 1120 DNA Artificial Sequence Description of Artificial Sequence Primer 11ggaacagctg atgaagatcc 20 12 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 12 ggtgagcaag gttacttcac 20

What is claimed is:
 1. A protein having following properties: (1) having hemolytic activity; (2) having a molecular weight of about 50,000 Da (determined by SDS gel electrophoresis); and (3) having the amino acid sequence represented by any of SEQ ID NO 1 and SEQ ID NO 2 as a partial amino acid sequence.
 2. The protein according to claim 1, wherein the protein is obtained from nematocyst of Carybdea alata.
 3. A protein having the hemolytic activity which has the same amino acid sequence as the hemolytic active protein according to claim 1, or the amino acid sequence modified by the addition and deletion of one or more amino acid, and/or the substitution by other amino acid to said amino acid sequence, and which is obtained from the cultivated product of the transformed cell prepared by genetic recombinant technique.
 4. A protein having amino acid sequence represented by SEQ ID NO 3, or the amino acid sequence modified by the addition and deletion of one or more amino acid, and/or the substitution by other amino acid to said amino acid sequence, and having hemolytic activity.
 5. The protein having hemolytic activity according to claim 3 or 4, wherein said protein is obtained from cultivated solution of transformed cell prepared by genetic recombinant technique using polynucleotide which hybridizes with polynucleotide encoding at least one of the amino acid sequences represented by SEQ ID NO 1 and SEQ ID NO
 2. 6. A process for preparing the protein according to claims 1, 2, or 4 comprising of ultrasonicating the nematocyst of Carybdea alata in phosphoric acid buffer solution, and extracting and purifying the supernatant fluid after centrifugation by ion exchange high performance liquid chromatography and gel filtration high performance liquid chromatography to obtain the protein.
 7. A process for preparing the protein according to claim 6, characterized by carrying out the ultrasonication for a nematocyst in phosphoric acid buffer solution, or treating by ion exchange high performance liquid chromatography and gel filtration high performance liquid chromatography in 10 mM phosphoric acid buffer solution (pH6.0) containing not less than 0.1M NaCl at not more than 10° C.
 8. A gene encoding the amino acid sequence of the protein having a hemolytic activity according to any one of claims 1 to
 5. 9. A vector comprising the gene according to claim
 8. 10. A host cell transformed by the vector as claimed in claim
 9. 11. A process for preparing a protein having hemolytic activity comprising culturing or growing a host cell as claimed in claim 10, and recovering the protein from said host cell or culture solution.
 12. A pharmaceutical composition comprising the protein according to any one of claims 1 to 5 as an active component.
 13. The pharmaceutical composition according to claim 12, wherein the composition has platelet agglutination effect.
 14. An antibody whose antigens are protein according to any one of claims 1 to 5, or those partial peptides.
 15. A pharmaceutical composition using the antibody according to claim
 14. 16. A pesticide comprising the protein according to any one of claims 1 to 5 as an active component. 